Reversing intestinal inflammation by inhibiting retinoic acid metabolism

ABSTRACT

An agent that increases local concentration of retinoic acid (RA) in the intestine through modifying enzymatic pathways involved in RA metabolism is administered in a dose effective to inhibit or reverse production of inflammatory mediators by intestinal dendritic cells and thereby reduce intestinal inflammation and tumor growth associated with intestinal inflammation.

CROSS REFERENCE

This application claims benefit and is a Continuation of applicationSer. No. 14/962,782 filed Dec. 8, 2015, which claims the benefit and isa Continuation of application Ser. No. 13/592,180, filed Aug. 22, 2012,now U.S. Pat. No. 9,248,120, issued Feb. 2, 2016, which claims benefitof U.S. Provisional Patent Application No. 61/526,574, filed Aug. 23,2011, which applications are incorporated herein by reference in theirentirety.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contract CA141468awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Pathological inflammation is emerging as an underlying mechanism fornumerous diseases. For example, the major forms of idiopathic IBD,ulcerative colitis and Crohn's disease are chronic inflammatorydisorders of the gastrointestinal tract. Animal studies have shown thatchronic intestinal inflammation precipitates as well as propagates tumorgrowth.

A number of sentinel cell populations in the intestinal mucosacontinuously monitor luminal microbes and other antigens. Among thesubsets of antigen-presenting cells, myeloid-derived dendritic cells area dominant subtype in the intestinal lamina propria and showconsiderable functional plasticity depending on the location, state ofmaturation, and stage of inflammation. Dendritic cells form an extensivenetwork beneath the intestinal epithelium and project long processesthrough the interstices of epithelial cells to sample luminal antigens.In response to TLR ligands, immature dendritic cells produce IL-23,which contributes to development of intestinal inflammation in murinemodels of colitis and intestinal inflammation. The remarkable capacityof dendritic cells to orchestrate distinct immune responses is aided bya panoply of environmental cues, which condition the cells to adoptspecific phenotypes in different settings. DCs in gut-associatedlymphoid tissue are of particular interest because they maintaintolerance to commensal flora as well as mount protective inflammatoryresponses in the face of pathogen incursion.

Migration of innate immune cells such as neutrophils, macrophages, anddendritic cells into target mucosal tissues depends on the expression ofcytokines, chemokines and adhesion molecules. Recruitment of activatedneutrophils, dendritic cells and macrophages into the lamina propria ingeneral amplifies the local immune response, whereas activated naturalkiller cell recruitment seems to enhance antimicrobial factors, leadingto attenuation of inflammation.

The CD4 T-cell lineage (T_(H)17) is characterized by the production ofIL-17. Its development is promoted by IL-23, in addition to IL-6 andTGFbeta. Evidence indicates that RORct (retinoic acid-related orphannuclear hormone receptor gamma-t) is necessary for T_(H)17 commitmentand differentiation. In addition to its ability to support thedevelopment of T_(H)17 cells, IL-23 induces the secretion of IL-17 bynon-T-cells in an inflammatory environment, and both T cells andmonocytes serve as sources of increased expression in the mucosa of IBDpatients.

An understanding and manipulation of inflammatory cells in the gut is ofgreat interest. The present invention addresses this issue.

SUMMARY OF THE INVENTION

Methods are provided for reducing intestinal inflammation, particularlychronic inflammation, and tumor growth precipitated by intestinalinflammation. In the methods of the invention, an effective dose of anagent is provided to the individual, where the agent increases localconcentration of retinoic acid (RA) in the intestine through modifyingenzymatic pathways involved in RA metabolism.

In some embodiments, an inhibitor of CYP26A1 is administered in a doseeffective form to neutralize a pre-existing inflammatory environment,prevent the development of inflammation, or maintain the tolerogenicfunctions of intestinal dendritic cells that maintain intestinaltolerance by inducing Treg formation. Alternatively, an agent thatincreases activity of retinaldehyde dehydrogenase or retinoldehydrogenase may be administered in a dose effective to neutralize orprevent inflammation in the intestine. The appropriate dose may bedetermined by evaluating the effect of the agent on functions ofintestinal dendritic cells, for example by monitoring synthesis ofcytokines such as IL-23 and IL-17, or by overall analysis of intestinalinflammation. The methods of the invention exclude administration ofretinoic acid directly.

Dendritic cells are shown to be reprogrammed due to local loss of thevitamin A metabolite, retinoic acid (RA), which occurs as a result ofinsufficient retinaldehyde dehydrogenase and an overabundance of thetranscriptional co-repressor, Ctbp1. The reprogrammed dendritic cellssecrete mainly pro-inflammatory cytokines and induce Th17 formation.Treatment by the methods of the invention replenish RA and neutralizethe inflammatory phenotype of the dendritic cells.

Other aspects and features will be readily apparent to the ordinarilyskilled artisan upon reading the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

The invention is best understood from the following detailed descriptionof exemplary embodiments when read in conjunction with the accompanyingdrawings. It is emphasized that, according to common practice, thevarious features of the drawings are not necessarily to-scale. On thecontrary, the dimensions of the various features are arbitrarilyexpanded or reduced for clarity. Included in the drawings are thefollowing figures:

FIG. 1A-1E. APC^(Min/+) SI-LPDCs accumulate over time and secrete morepro-inflammatory cytokines compared to WT SI-LPDCs upon TLR stimulation.DCs were defined as PI− EpCAM− CD45+ DX5− CD3e− CD19− CD11c^(hi) MHCII+in all the tissues.

WT;

APC^(Min/+) (FIG. 1A) A representative contour plot depicting how DCsare gated by CD11c and MHC II expression. (FIG. 1B) A bar graph of themean frequency (with SEM) of CD11c+MHCII+ DCs as a percentage of PI−Lineage− cells. (FIG. 1C) Representative contour plots of SPL, mLN/PPand SI-LP DCs divided by CD11 b and CD103 expression. Below each FACSplot are bar graphs showing the mean frequency (with SEM) of each subsetin that tissue as a percentage of total CD11c+ MHCII+ DCs. (FIG. 1D)Mean frequency (with SEM) of cells expressing the co-stimulatorymolecules CD40, CD80, CD83, CD86 and PDL1 as a percentage of thedifferent CD11 b CD103 subsets. Data for (FIG. 1A-1D) were obtained atintermediate stage disease. Inset values in the contour plots are themean frequencies of each population pooled using at least 4 mice perstrain (WT and APC^(Min/+)) per experiment, from 5 independentexperiments. (FIG. 1E) 5×10⁴ FACS-purified WT and APC^(Min/+) SI-LPDCswere stimulated with a panel of 6 different TLR agonists—Pam3Csk4 (1μg/ml), Poly I:C (10 μg/ml), LPS (10 μg/ml), flagellin (1 μg/ml), R848(10 μg/ml), CpG 2336 (10 μg/ml). Supernatants were collected after 48hours. Representative bar graphs show mean production (with SEM) of thecytokines IL-6, TNFα and IL-2p40 as measured by standard ELISA. Data in(FIG. 1E) was obtained at late stage of disease. Data are representativeof 4 independent experiments, with DCs pooled from 8 mice per strain (WTand APC^(Min/+)) per experiment. An unpaired student's t test with 95%confidence interval was performed. P<0.05=*; p<0.001=**; p<0.0001=***.

FIG. 2A-2G. APC^(Min/+) SI-LPDCs have a reduced capacity to induceFoxp3+T_(Regs) and IL-10 producing CD4+ T cells, but instead generateTh17 cells, in an RA-dependent manner. 2×10⁴ of sorted CD11c^(hi) MHCII⁺DCs were co-cultured for 5 days with 1×10⁵ MACS-enriched CD4+CD62L+Foxp3− naïve T cells from the spleen and lymph nodes of OT-IITCR-transgenic mice, along with OVA₃₂₃₋₃₃₉ peptide and 10 ng/mlrecombinant human TGF-β. 5 ng/ml of recombinant human IL-2 was added tothe cultures every other day beginning on day 2.

WT;

APC^(Min/+) (FIG. 2A) DCs used in these cultures were purified from SPL,mLN/PP and SI-LP, and stimulated with 5 nM OVA₂₃₃₋₃₃₉. Representativebar graph shows the mean frequency (with SEM) of Foxp3 induction from 9independent experiments, with DCs pooled from at least 5 WT and at least3 APC^(Min/+) mice per experiment. (FIG. 2B) SI-LPDCs were used in theseco-cultures, and stimulated with 200 nM, 1 μM and 5 μM OVA₃₂₃₋₃₃₉.Representative bar graph shows the mean frequency (with SEM) of Foxp3induction from 4 independent experiments, with DCs pooled from at least5 WT and at least 3 APC^(Min/+) mice per experiment. (FIG. 2C) CD11c+MHCII+ SI-LP DCs were further sorted into the CD103+ and CD103− subsetsand cultured with naïve CD4 T cells as before. Bar graph shows the meanfrequency (with SEM) of Foxp3 induction in a representative experimentof 2 independent experiments, with DCs pooled from at least 5 WT and atleast 3 APC^(Min/+) mice per experiment. (FIG. 2D) In these experiments,10 nM all-trans RA or 1 μM of the RAR antagonist LE540 was added towhole SI-LPDC-T cell co-culture wells, in addition to TGF-β1. Cells werere-stimulated on day 4 with plate-bound anti-CD3 and anti-CD28 (1 μg/mleach) for 18 hours. Supernatants were collected on day 5 to assay forcytokine production by standard ELISA. Representative bar graphs showthe mean production (with SEM) of IL10 (FIG. 2E), IL17A (FIG. 2F) andIL6 (FIG. 2G). Data in this figure were obtained at late stage disease,and are representative of 4 independent experiments, with DCs pooledfrom at least 5 WT and at least 3 APC^(Min/+) mice per experiment. Anunpaired student's t test with 95% confidence interval was performed.P<0.05=*; p<0.001=**; p<0.0001=***.

FIG. 3A-3C. APC^(Min/+) SI-LP DCs and IECs have lower expression ofRaldhs than WT, resulting in loss of RA in the tumor milieu.

WT;

APC^(Min/+). Total RNA from FACS-purified SI-LP DCs, splenic DCs (FIG.3A) and epithelial cells (IECs) (FIG. 3B) was extracted and assayed intriplicate by qPCR for the genes Raldh1a1, Raldh1a2, Raldh1a3 and Ctbp1(FIG. 3C). Each sample was normalized to ubiquitin b expression.Representative bar graphs show mean relative expression (with SEM) oftriplicate samples. Data are representative of 3 independent qPCRexperiments, with DCs and IECs pooled from 3 sorts per timepoint, usingat least 5 WT and at least 3 APC^(Min/+) mice per sort. An unpairedstudent's t test with 95% confidence interval was performed. P<0.05=*;p<0.001=**; p<0.0001=***.

FIG. 4A-4F. FAP adenoma tissue exhibits IL-17-driven inflammation andloss of RA. Normal colon, FAP adenoma and sessile serrated polyp (SSP)sections were stained by immunohistochemistry for IL-17A (FIG. 4A) orco-stained by immunofluorescence with DC-SIGN and β-catenin (FIG. 4B),Ctbp1 (FIG. 4C), Raldh1a1 (FIG. 4D), Raldh1a2 (FIG. 4E), CYP26A1 (FIG.4F). Shown are samples from one representative normal colon, tworepresentative FAP adenoma and one representative SSP patient. Imagesfor FAP P1 show serial sections of the same polyp with underlyingadjacent normal grossly uninvolved tissue. Overall, samples from a totalof 11 normal colon, 8 FAP adenoma, 2 FAP adenocarcinoma and 4 SSPpatients were analyzed. All images were captured using the same exposuretime and the same brightfield or fluorescence settings for each proteinof interest. White magnification bars in (FIG. 4A-4F) are 100 μM.

FIG. 5A-5I. A vitamin A-deficient diet exacerbates disease inAPC^(Min/+) mice (FIG. 5A-5D) 10 week old WT (open circles) andAPC^(Min/+) (filled triangles) mice were placed on 1500 ppm Celecoxib (

,

); 100 ppm Rosiglitazone (

,

); a vitamin A deficient diet (0 IU/g of vitamin A) (

,

); or a base diet (4 IU/g of vitamin A) (

,

) for 10 consecutive weeks. Mice were monitored for disease progressionon the basis of hematocrit and body weight change. Line graphs showKaplan-Meier survival curves (FIG. 5A), mean change in percentage bodyweight over original body weight (FIG. 5B) and hematocrits (FIG. 5C),with SEMs. 5 mice were used per strain for each diet. Data arerepresentative of 2 independent experiments. Liarozole, a CYP26A1inhibitor, ameliorates disease in APC^(Min/+) mice by increasing localRA which reverses the SI-LPDC pro-inflammatory phenotype. 8 week old WTand APC^(Min/+) mice were placed on 40 ppm of Liarozole for 6consecutive weeks. APC^(Min/+)—Base diet

; APC^(Min/+)—Liarozole

; WT—Liarozole

. Disease was monitored as described in (FIG. 5A-5D). In theseexperiments, small intestinal polyps were enumerated at 14 weeks, thepoint of euthanasia. A scatter plot shows the mean number of polyps(with SEM) (FIG. 5H) between APC^(Min/+) mice on Liarozole compared tobase diet. 5 mice were used per strain in each diet. Data arerepresentative of 2 independent experiments. Significance in diseaseindicators of the different diets was calculated by comparing values tothose obtained on the base diet. For calculating significance in DAI, aWlcoxon-rank-sum test was used. (FIG. 5I) All-trans retinoic acid (RA)was extracted from the duodenum, jejunum, ileum, colon and eye andquantified by tandem mass spectrometry. WT—base diet

; APC^(Min/+)—Base diet

; APC^(Min/+)—Liarozole

. Bar graphs show mean amounts of retinoic acid per gram tissue (withSEM) among mice of the same strain. 5 WT, 5 APC^(Min/+) and 4Liarozole-treated APC^(Min/+) mice were used in this experiment. Fordata in this figure, an unpaired student's t test with 95% confidenceinterval was performed unless otherwise stated. P<0.05=*; p<0.001=**;p<0.0001=***. 8 week-old WT and APC^(Min/+) mice were placed on 40 ppmof Liarozole for 6 consecutive weeks as described in FIGS. 5A-5I.WT—base diet

; APC^(Min/+)—Base diet

; APC^(Min/+)—Liarozole

. (FIG. 5A) At the 14 week time point of euthanasia, 2×10⁴ FACS-purifiedWT and APC^(Min/+) SI-LPDCs were stimulated with Pam3Csk4 (1 μg/ml), LPS(10 μg/ml) and R848 (10 μg/ml). Supernatants were collected after 48hours. Representative bar graphs show mean production (with SEM) of thecytokines IL-6, TNFα and IL-2p40 as measured by standard ELISA. Data arerepresentative of 2 independent experiments, with at least 2 mice perstrain. (FIG. 5B) A T_(Reg) induction assay was performed as describedin FIG. 2 on SI-LPDCs sorted from these mice. Representative bar graphshows the mean frequency (with SEM) of Foxp3 induction from 2independent experiments, with DCs pooled from at least 2 mice perstrain. (FIG. 5C) T cells in the SI-LPDC-T cell co-culture wasre-stimulated as described in FIG. 2 and assayed for IL10, IL17A and IL6by standard ELISA as before. For data in this figure, an unpairedstudent's t test with 95% confidence interval was performed. P<0.05=*;p<0.001=**; p<0.0001=***.

FIG. 6. Proposed Model: In the APC^(Min/+) mouse, the immune state ofthe intestine, departs from homeostatic tolerance to chronicinflammation. Truncated APC protein leads to an accumulation of thetranscription factor Ctbp1, which in turns suppresses retinoldehydrogenases in the epithelium and may lead to a reduction in localconcentrations of RA. Upregulation of Cox2 in the inflamed tissue leadsto greater production of PGE₂, which has been reported to suppressRaldh1a2 in DCs and likely does so to the surrounding epithelium aswell. Lack of RA in the intestinal milieu conditions or ‘reprograms’SI-LPDCs to a pro-inflammatory phenotype, in which IL6 productioncombined with TGFβ found locally, contributes to a deleterious Th17response that promotes tumor growth.

FIG. 7. Morphology of sorted SI-LPDCs from WT and APC^(Min/+) mice.Sorted PI-EpCAM− CD45+ DX5− CD3e− CD19− CD11c^(hi) MHCII+ LP DCs werestained with May Grumwald-Giemsa.

FIG. 8A-8C. APC^(Min/+) SI-LPDCs have a reduced capacity to induceFoxp3+T_(Regs) in a tissue-specific, dose-dependent and time-dependentmanner. A T_(Reg) induction assay was performed as described in FIG. 2.(FIG. 8A-8C) Representative dot plots of Foxp3 versus CFSE in Thy1.2+CD4+ cells. The inset values of the bordered population show the meanfrequency of Foxp3+ cells as a percentage of Thy1.2+ CD4+ cells. (FIG.8A) DCs used in these cultures were purified from SPL, mLN/PP and SI-LP.(FIG. 8B) SI-LPDCs were incubated with 200 nM, 1 μM and 5 μM OVA₃₂₃₋₃₃₉.(FIG. 8C) SI-LPDCs were isolated from early, intermediate and late stagedisease.

FIG. 9A-9B. ADH class I and II expression in APC^(Min/+) IECs andSI-LPDCs increased at intermediate stage and decreased at subsequentlate stage compared to WT counterparts. RT-qPCR on FACS-purified WT andAPC^(Min/+) SI-LP DCs, splenic DCs (FIG. 9A) and epithelial cells (IECs)(FIG. 9B) was performed as described in FIG. 3.

WT

; APC^(Min/+) Representative bar graphs show mean relative expression(with SEM) of triplicate samples. Data are representative of 3independent qPCR experiments, with DCs and IECs pooled from 3 sorts pertime point, using at least 5 WT and at least 3 APC^(Min/+) mice persort. Shown is the ADH class I and II expression in DCs (a) and in IECs(FIG. 9B).

FIG. 10A-10B. Quantification of retinyl esters and retinol in vivo.Retinyl esters (RE) and all-trans retinol (ROL) were extracted from theduodenum, jejunum, ileum, colon and eye and quantified by HPLC. Bargraphs show mean amounts of retinoid per gram tissue (with SEM) amongmice of the same strain. 5 WT, 5 APC^(Min/+) and 4 Liarozole-treatedAPC^(Min/+) mice were used in this experiment. An unpaired student's ttest with 95% confidence interval was performed. P<0.05=*; p<0.001=**;p<0.0001=***.

FIG. 11. Isotype control for immunohistochemistry andimmunofluorescence. Normal colon sections were stained with an isotypecontrol (Rabbit IgG) to the rabbit primaries used against β-catenin,Ctbp1, Raldh1a1. Raldh1a2 and CYP26A1.

FIG. 12. Wildtype mice on base, rosiglitazone, celecoxib and vitaminA-deficient diets gain in weight comparably. Wildtype mice on thevarious diets 10 week-old WT mice were placed on 1500 ppm Celecoxib, 100ppm Rosiglitazone, a vitamin A deficient diet (0 IU/g of vitamin A), oron a base diet (4 IU/g of vitamin A), for 10 consecutive weeks. Linegraph show mean change in percentage body weight over original bodyweight. Data are representative of 2 independent experiments.

FIG. 13. APC^(Min/+) SI-LPDCs display higher expression of Cox2 comparedto WT SI-LPDCs. RT-qPCR on FACS-purified WT and APC^(Min/+) SI-LP DCswas performed as described in FIG. 3.

WT;

APC^(Min/+) Representative bar graph shows mean relative expression(with SEM) of triplicate samples. Data is from 1 independent qPCRexperiment, with DCs and pooled from 3, using at least 5 WT and at least3 APC^(Min/+) mice per sort.

FIG. 14A-14F. Direct supplementation of RA does not affect tumorfrequency or intestinal RA levels in APC^(Min/+) mice, but Liarozole, aCYP26A1 inhibitor, improves all tested disease parameters. Groups of 8week-old APC^(Min/+) mice were injected i.p. with or without 200μg/mouse RA in sunflower oil injected twice weekly while on a base diet,or placed on 40 ppm of Liarozole for 6 consecutive weeks. APC^(Min/+) onBase diet/; APC^(Min/+) on Liarozole/; APC^(Min/+) given RA i.p./; WT onLiarozole. (FIG. 14A) Small intestine tumors were enumerated at 14weeks. Scatter plot shows the mean number of tumors (with SEM) inAPC^(Min/+) mice on base, Liarozole or RA i.p. (a). Line graphs showmean change in percentage body weight (FIG. 14B) and hematocrit (FIG.14C). For (FIG. 14A-14C), data for mice on base diet and Liarozole areaggregated from 4 independent experiments, with at least 4 mice pergroup, while data for RA i.p.-injected mice are aggregated from 2independent experiments, with at least 3 mice per group. (FIG. 14D)Concomitant to experiments performed in FIG. 4D, RA was quantified byLC/MS in 5 Liarozole treated and 5 RA i.p.-injected APC^(Min/+) mice.Shown are mean RA levels (with SEM) in each tissue isolated at the 14week time point. (FIG. 14E) Dot plots show the mean frequency of IL-17A-and IFNγ-producing CD4⁺ T cells in freshly-isolated SI-LP from WT,APC^(Min/+) and Liarozole-treated APC^(Min/+) mice. Also shown are bargraphs depicting mean frequency (with SEM) of IL-17A- and IFNγ-producingCD4+ T cells from these mice. (FIG. 14F) CD103− SI-LPDCs from WT,APC^(Min/+) and Liarozole-treated APC^(Min/+) mice were co-cultured inthe T cell differentiation assays performed as in FIG. 1A-1E. Insetvalues on the representative dot plots show the mean frequency of IL-10-and IL-17A− producing CD4⁺ T cells induced. In (ef) IL-17A⁺, IFNγ⁺, orIL-10⁺ cells are shown as a percentage of CD4⁺ T cells. Data in (FIG.14E-14F) are from 2 independent experiments, with at least 4 mice pergroup. DCs obtained in (FIG. 14F) were pooled from all mice in the samegroup in each experiment. P<0.05=*; p<0.001=**; p<0.0001=***.

FIG. 15. Talarozole, a highly specific inhibitor of CYP26A1, amelioratesdisease in APC^(Min/+) mice. 8 week-old APC^(Min/+) mice were placed on8 ppm of Talarozole for 6 consecutive weeks. APC^(Min/+)—Base diet;APC^(Min/+)—Talarozole. At 14 weeks, tumors in the small and largeintestine were enumerated. Results shown are aggregated from 2independent experiments, with 5 mice per group.

FIG. 16. Paraffin embedded normal, inflamed, dysplastic or carcinomacolon sections were stained by immunofluorescence for CYP26A1 (in red)and DC-SIGN (in green). Shown are samples from 2 different patients.Patient 1: Normal, Dysplastic and Carcinoma sections from a colectomysample and Patient 2 Inflamed and dysplastic regions from a colectomysample. All images were captured using the same exposure time and thesame brightfield or fluorescence settings for each protein of interest.Similar results were obtained from staining with Raldh1a1 and Raldh1a2.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It is to be understood that the invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, exemplary methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited. Itis understood that the present disclosure supercedes any disclosure ofan incorporated publication to the extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “thepolypeptide” includes reference to one or more polypeptides andequivalents thereof known to those skilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude anyelement which may be optional. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the disorderbeing treated and the general state of the patient's own immune system.

“Polypeptide” and “protein” as used interchangeably herein, canencompass peptides and oligopeptides. Where “polypeptide” is recitedherein to refer to an amino acid sequence of a naturally-occurringprotein molecule, “polypeptide” and like terms are not necessarilylimited to the amino acid sequence to the complete, native amino acidsequence associated with the recited protein molecule, but instead canencompass biologically active variants or fragments, includingpolypeptides having substantial sequence similarity or sequence identifyrelative to the amino acid sequences provided herein. In general,fragments or variants retain a biological activity of the parentpolypeptide from which their sequence is derived.

As used herein, “polypeptide” refers to an amino acid sequence of arecombinant or non-recombinant polypeptide having an amino acid sequenceof i) a native polypeptide, ii) a biologically active fragment of anpolypeptide, or iii) a biologically active variant of an polypeptide.Polypeptides suitable for use can be obtained from any species, e.g.,mammalian or non-mammalian (e.g., reptiles, amphibians, avian (e.g.,chicken)), particularly mammalian, including human, rodenti (e.g.,murine or rat), bovine, ovine, porcine, murine, or equine, particularlyrat or human, from any source whether natural, synthetic, semi-syntheticor recombinant. In general, polypeptides comprising a sequence of ahuman polypeptide are of particular interest.

The term “derived from” indicates molecule that is obtained directlyfrom the indicated source (e.g., when a protein directly purified from acell, the protein is “derived from” the cell) or information is obtainedfrom the source, e.g. nucleotide or amino acid sequence, from which themolecule can be synthesized from materials other than the source ofinformation.

The term “isolated” indicates that the recited material (e.g,polypeptide, nucleic acid, etc.) is substantially separated from, orenriched relative to, other materials with which it occurs in nature(e.g., in a cell). A material (e.g., polypeptide, nucleic acid, etc.)that is isolated constitutes at least about 0.1%, at least about 0.5%,at least about 1% or at least about 5% by weight of the total materialof the same type (e.g., total protein, total nucleic acid) in a givensample.

The terms “subject” and “patient” are used interchangeably herein tomean a member or members of any mammalian or non-mammalian species thatmay have a need for the pharmaceutical methods, compositions andtreatments described herein. Subjects and patients thus include, withoutlimitation, primate (including humans), canine, feline, ungulate (e.g.,equine, bovine, swine (e.g., pig)), avian, and other subjects. Humansand non-human animals having commercial importance (e.g., livestock anddomesticated animals) are of particular interest. As will be evidencefrom the context in which the term is used, subject and patient refer toa subject or patient susceptible to infection by a Flaviviridae virus,particularly HCV.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compoundscalculated in an amount sufficient to produce the desired effect inassociation with a pharmaceutically acceptable diluent, carrier orvehicle. The specifications for the novel unit dosage forms depend onthe particular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, dileuent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, dileuent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” issterile, and is usually free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal and the like.

T helper 17 cells (Th17) are a subset of T helper cells, characterizedby their production of interleukin 17 (IL-17). They are considereddevelopmentally distinct from Th1 and Th2 cells and excessive amounts ofthe cell are thought to play a key role in autoimmune disease. Inhumans, a combination of TGF-β, IL-1β and IL-23 induces Th17differentiation from naive T cells. Both interferon gamma (IFNγ) andIL-4, the main stimulators of Th1 and Th2 differentiation respectively,negatively regulate Th17 differentiation.

T helper 1 cells (Th1). Proliferating helper T cells that develop intoeffector T cells differentiate into two major subtypes of cells known asTh1 and Th2 cells. Th1 cells primarily produce IFN-γ and TNF-βcytokines. IFN-γ increases the production of interleukin-12 by dendriticcells and macrophages, and via positive feedback, IL-12 stimulates theproduction of IFN-γ in helper T cells, thereby promoting the Th1profile. IFN-γ also inhibits the production of cytokines such as IL-4.Conditions that polarize to the T_(H)1 type include antigen presentingcells and IL-12.

Interleukin-17 (IL-17) refers to a group of cytokines called the IL-17family. IL-17 shows high homology to viral IL-17 encoded by an openreading frame of the T lymphotropic rhadinovirus Herpesvirus saimiri. Toelicit its functions, IL-17 binds to a type I cell surface receptorcalled IL-17R of which there are at least three variants IL17RA, IL17RB,and IL17RC. Members of the IL-17 family include IL-17B, IL-17C, IL-17D,IL-17E (also called IL-25), and IL-17F. All members of the IL-17 familyhave a similar protein structure, with four highly conserved cysteineresidues critical to their 3-dimensional shape, although with nosequence similarity to any other known cytokines. Numerous immuneregulatory functions have been reported for the IL-17 family.

IL-23 alpha subunit is a protein encoded by the IL23A gene (see Oppmannet al. (2001) Immunity 13 (5): 715-25). This gene encodes the p19subunit of the heterodimeric cytokine interleukin 23 (IL23). IL23 iscomposed of this protein and the p40 subunit of interleukin 12. Thereceptor of IL23 is formed by the beta 1 subunit of IL12 (IL12RB1) andan IL23 specific subunit, IL23R. Both IL23 and IL12 can activate thetranscription activator STAT4, and stimulate the production ofinterferon-gamma (IFNG). In contrast to IL12, which acts mainly on naiveCD4(+) T cells, IL23 preferentially acts on memory CD4(+) T cells.

Dendritic cell. As used herein, the term refers to any member of adiverse population of morphologically similar cell types found inlymphoid or non-lymphoid tissues. Dendritic cells are a class of“professional” antigen presenting cells, and have a high capacity forsensitizing MHC-restricted T cells. Dendritic cells may be recognized byfunction, or by phenotype, particularly by cell surface phenotype. Thesecells are characterized by their distinctive morphology, intermediate tohigh levels of surface MHC-class II expression and ability to presentantigen to T cells, particularly to naive T cells (Steinman et al.(1991) Ann. Rev. Immunol. 9:271; incorporated herein by reference forits description of such cells).

The vitamin A metabolite all-trans-retinoic acid (RA) is an essentialsignaling molecule in embryonic development and throughout life; apotent regulator of cell differentiation, proliferation, and apoptosisin various cell types. RA acts through specific RA nuclear receptors(RARα, β, and γ) and their heterodimeric counterparts, theretinoid-X-receptors (α, β, and γ) to positively or negatively regulateexpression of RA target genes by binding to their respective responseelements. Vitamin A deficiency has been linked to increasedsusceptibility to carcinogenesis in animal models. Although essentiallyall cell types express nuclear retinoic acid receptors, cellularresponsiveness is determined by RA bioavailability regulated by thecoordinated balance between vitamin A nutritional status and RAbiosynthesis and catabolism. The RA-metabolizing cytochrome P450sCYP26A1, B1, and C1 convert RA into rapidly excreted oxoderivatives(4-OH RA, 4-oxo RA, 18-OH RA), while retinaldehyde dehydrogenasegenerates RA. Inhibitors of the CYP26 gene family are of interest foruse in the methods of the invention, including without limitationCYP26A1.

CYP26A1 is a member of the cytochrome P450 superfamily of enzymes. Thecytochrome P450 proteins are monooxygenases. This endoplasmic reticulumprotein acts on retinoids, including all-trans-retinoic acid (RA), withboth 4-hydroxylation and 18-hydroxylation activities. Two alternativelyspliced transcript variants of this gene, which encode the distinctisoforms, have been reported.

Inhibitors of CYP26A1 can increase local concentrations of RA. Forexample, an orally administered inhibitor, particularly a formulationthat provides for enteric delivery, can raise the RA concentration inintestinal tissues. Inhibitors of CYP26A1 are known in the art, andinclude, without limitation, talarozole, ketoconazole; liarozole;[S—(R*,R*)]—N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine(R116010);(R)—N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine(R115866); the retinoic acid receptor (RAR)γ agonist CD1530; the pan-RARagonist4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid; peroxisome proliferator-activated receptor ligands rosiglitazoneand pioglitazone; etc.

Aldehyde dehydrogenase 1 (retinal dehydrogenase, RALDH1) is a livercytosolic isoform of acetaldehyde dehydrogenase. Retinaldehyde isgenerated by ADH1 from retinol, and its concentration is determined inlarge part by its subsequent catabolism by RALDH1 to retinoic acid, seeHempel et al. (1984) Europ. J. Biochem. 141: 21-35; Hsu et al. (1985)Proc. Nat. Acad. Sci. 82: 3771-3775. Agonists of RALDH1, or other agentsthat increase activity of RALDH1 are of interest for the methods of theinvention.

Retinol dehydrogenase 10 (RDH10) generates all-trans retinal fromall-trans retinol and may plan an important role in the photic visualcycle. The first oxidative step of vitamin A metabolism is catalyzed inlarge part by RDH10 and is critical for spatiotemporal synthesis ofretinoic acid, Sandell et al. (2007) Genes Dev. 21: 1113-1124; Wu et al.(2002) Invest. Ophthal. Vis. Sci. 43: 3365-3372. Agonists of RDH10, orother agents that increase activity of RDH10 are of interest for themethods of the invention.

The term colorectal cancer includes all cancers of the colon and/orrectum, but particularly adenocarcinoma of the colon (e.g., mucinous(colloid) adenocarcinoma or signet ring adenocarcinoma). Other types ofcolorectal cancer included by the term include the following varietiesof colon cancer: neuroendocrine, lymphoma, melanoma, squamous cell,sarcoma and carcinoid. The term colorectal cancer also includes allstages of colorectal cancer; for example, under the Modified DukeStaging System or TNM system (Tumor, Node, Metastasis). The stagesassociated with these systems are well known by practitioners ofordinary skill in the art.

In the methods of the invention, the agent(s) may be administered to asubject to treat an inflammatory condition, which inflammation maypredispose to colorectal cancer, and may include individuals havingfamilial adenomatous polyposis (FAP), hereditary nonpolyposis coloncancer (HNPCC) (i.e., Lynch I Syndrome or Lynch II Syndrome),inflammatory bowel disease, such as chronic ulcerative colitis (UC) orCrohn's disease, other family cancer syndromes (e.g., Peutz-JegherSyndromem and Familial Juvenile Polyposis), adenomatous polyps (e.g.,sessile (flat with a broad base and no stalk); tubular (composed oftubular glands extending downward from the outer surface of the polyp);villous (composed of fingerlike epithelial projections extending outwardfrom the surface of the bowel mucosa); pedunculated (attached by anarrow base and a long stalk), and sporadic forms of colon cancer.

Familial adenomatous polyposis (FAP) is an inherited condition in whichnumerous polyps form mainly in the epithelium of the large intestine. Ingeneral, while these polyps start out benign, malignant transformationinto colon cancer occurs when not treated. Familial juvenile polyposis(FJP) is an autosomal dominant condition characterized by multiplejuvenile polyps of the gastrointestinal (GI) tract. Kindreds have beendescribed in which there is involvement of the colon only, the upper GItract or both upper and lower GI tracts. FJP is a hamartomatouspolyposis syndrome. Adenomatous polyps (adenomas) of the colon andrectum may be benign (noncancerous) growths that may be precursorlesions to colorectal cancer. In general, polyps greater than onecentimeter in diameter are associated with a greater risk of cancer. Ifpolyps are not removed, they typically continue to grow and can becomecancerous.

Crohn's Disease (Regional Enteritis; Granulomatous Ileitis orIleocolitis) is a chronic transmural inflammatory disease that usuallyaffects the distal ileum and colon but may occur in any part of the GItract. Symptoms include diarrhea and abdominal pain. Abscesses, internaland external fistulas, and bowel obstruction may arise. Extraintestinalsymptoms, particularly arthritis, may occur. Diagnosis is by colonoscopyand barium contrast studies. The most common initial presentation ischronic diarrhea with abdominal pain, fever, anorexia, and weight loss.The abdomen is tender, and a mass or fullness may be palpable. Grossrectal bleeding is unusual except in isolated colonic disease, which maymanifest similarly to ulcerative colitis. Some patients present with anacute abdomen that simulates acute appendicitis or intestinalobstruction. About 33% of patients have perianal disease (especiallyfissures and fistulas), which is sometimes the most prominent or eveninitial complaint. In children, extraintestinal manifestationsfrequently predominate over GI symptoms; arthritis, fever of unknownorigin, anemia, or growth retardation may be a presenting symptom,whereas abdominal pain or diarrhea may be absent.

Established Crohn's disease is rarely cured but is characterized byintermittent exacerbations and remissions. Some patients suffer severedisease with frequent, debilitating periods of pain. However, withjudicious medical therapy and, where appropriate, surgical therapy, mostpatients function well and adapt successfully. Disease-related mortalityis very low. GI cancer, including cancer of the colon and small bowel,is the leading cause of excess Crohn's disease-related mortality.

Irritable bowel syndrome consists of recurring upper and lower GIsymptoms, including variable degrees of abdominal pain, constipation ordiarrhea, and abdominal bloating. Diagnosis is clinical. Treatment isgenerally symptomatic, consisting of dietary management and drugs,including anticholinergics and agents active at serotonin receptors.There are no consistent motility abnormalities. Some patients have anabnormal gastro-colonic reflex, with delayed, prolonged colonicactivity. There may be reduced gastric emptying or disordered jejunalmotility. Some patients have no demonstrable abnormalities, and in thosethat do, the abnormalities may not correlate with symptoms. Small-boweltransit varies: sometimes the proximal small bowel appears to behyperreactive to food or parasympathomimetic drugs. Intraluminalpressure studies of the sigmoid show that functional constipation canoccur with hyperreactive haustral segmentation (ie, increased frequencyand amplitude of contractions). In contrast, diarrhea is associated withdiminished motor function. Thus strong contractions can, at times,accelerate or delay transit.

Diagnosis is based on characteristic bowel patterns, time and characterof pain, and exclusion of other disease processes through physicalexamination and routine diagnostic tests. Diagnostic testing should bemore intensive when “red flags” are present: older age, weight loss,rectal bleeding, vomiting. Proctosigmoidoscopy with a flexiblefiberoptic instrument should be performed. Introduction of thesigmoidoscope and air insufflation frequently trigger bowel spasm andpain. The mucosal and vascular patterns in IBS usually appear normal.Colonoscopy is preferred for patients>40 with a change in bowel habits,particularly those with no previous IBS symptoms, to exclude colonicpolyps and tumors. In patients with chronic diarrhea, particularly olderwomen, mucosal biopsy can rule out possible microscopic colitis.

Methods of the Invention

Methods are provided for reducing intestinal inflammation, particularlychronic inflammation, and tumor growth precipitated by intestinalinflammation. In the methods of the invention, an effective dose of anagent is provided to the individual, where the agent increases localconcentration of retinoic acid (RA) in the intestine through modifyingenzymatic pathways involved in RA metabolism. In particular, aninhibitor of a CYP26, enzyme, for example CYP26A1, may be administeredin a dose effective to neutralize an inflammatory environment and/ormaintain the tolerogenic functions of intestinal dendritic cells thatmaintain intestinal tolerance by inducing Treg formation. Alternatively,an agent that increases activity of retinaldehyde dehydrogenase orretinol dehydrogenase may be administered in a dose effective tomaintain the tolerogenic functions of dendritic cells. The appropriatedose may be determined by evaluating the effect of the agent onfunctions of dendritic cells, or by overall analysis of intestinalinflammation. The methods of the invention exclude administration ofretinoic acid directly. The administration of inhibitors of CYP26A1,including without limitation liarozole and talarozole, are of particularinterest.

The term “therapeutically effective amount” or “therapeuticallyeffective dosage” may mean that amount or dosage of an agent orcombination thereof of the invention or composition thereof that willelicit a biological or medical response of a tissue, system, patient,subject or host that is being sought by the administrator (such as aresearcher, doctor or veterinarian) which includes any measurablealleviation of the signs, symptoms and/or clinical indicia of intestinalinflammation, including colorectal cancer (e.g., tumor growth and/ormetastasis) including the prevention, slowing or halting of progressionof the inflammation to any degree whatsoever.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single dose may beadministered or several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies or the particular circumstances or requirements of thetherapeutic situation. For example, dosage may be determined oradjusted, by a practitioner of ordinary skill in the art (e.g.,physician or veterinarian) according to the patient's age, weight,height, past medical history, present medications and the potential forcross-reaction, allergies, sensitivities and adverse side-effects. Forexample, the physician or veterinarian could start doses of the agent atlevels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved. The effectiveness of a given dose or treatmentregimen of an agent of the invention can be determined, for example, bydetermining the concentration of pro-inflammatory cytokines such asIL-23; whether a tumor being treated in the subject shrinks or ceases togrow. The size and progress of a tumor can be easily determined, forexample, by X-ray, magnetic resonance imaging (MRI) or visually in asurgical procedure. In general, tumor size and proliferation can bemeasured by use of a thymidine PET scan (see e.g., Wells et al., Clin.Oncol. 8: 7-14 (1996)). Generally, the thymidine PET scan includes theinjection of a radioactive tracer, such as [2-¹¹C]-thymidine, followedby a PET scan of the patient's body (Vander Borght et al.,Gastroenterology 101: 794-799, 1991; Vander Borght et al., J. Radiat.Appl. Instrum. Part A, 42: 103-104 (1991)). Other tracers that can beused include [¹⁸F]-FDG (18-fluorodeoxyglucose), [¹²⁴I]IUdR(5-[¹²⁴I]iodo-2′-deoxyuridine), [⁷⁶Br]BrdUrd (Bromodeoxyuridine),[¹⁸F]FLT (3′-deoxy-3′ fluorothymidine) or [¹¹C]FMAU(2′-fluoro-5-methyl-1-β-D-arabinofuranosyluracil).

Methods for treating or preventing intestinal inflammation, includinginflammation leading to colorectal cancer by administering apharmaceutical composition comprising an agent that increases RA levelsby altering enzymatic activity involved in RA metabolism, in associationwith a pharmaceutically acceptable carrier are also within the scope ofthe present invention (e.g., in a single composition or separately in akit) as are combinations and compositions including such pharmaceuticalcompositions. The pharmaceutical compositions may be prepared by anymethods well known in the art of pharmacy; see, e.g., Gilman, et al.,(eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed.,Pergamon Press; A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18th Edition, (1990), Mack Publishing Co., Easton, Pa.; Avis, et al.,(eds.) (1993) Pharmaceutical Dosage Forms: Parenteral MedicationsDekker, N.Y.; Lieberman, et al., (eds.) (1990) Pharmaceutical DosageForms: Tablets Dekker, N.Y.; and Lieberman, et al., (eds.) (1990),Pharmaceutical Dosage Forms: Disperse Systems Dekker, N.Y.

A pharmaceutical composition can be prepared using conventionalpharmaceutically acceptable excipients and additives and conventionaltechniques. Such pharmaceutically acceptable excipients and additivesinclude non-toxic compatible fillers, binders, disintegrants, buffers,preservatives, anti-oxidants, lubricants, flavorings, thickeners,coloring agents, emulsifiers and the like. All routes of administrationare contemplated including, but not limited to, parenteral (e.g.,subcutaneous, intravenous, intraperitoneal, intramuscular, topical,intraperitoneal, inhalation, intra-cranial) and non-parenteral (e.g.,oral, transdermal, intranasal, intraocular, sublingual, rectal andtopical).

Oral administration is of interest, including enteric formulations,which may include acid stable agents that maintain activity undergastrointestinal conditions, enteric coatings of pills, and the like,where there is a significant activity of the agent in intestinaltissues.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Theinjectables, solutions and emulsions can also contain one or moreexcipients. Excipients include, for example, water, saline, dextrose,glycerol or ethanol. In addition, if desired, the pharmaceuticalcompositions to be administered may also contain minor amounts ofnon-toxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, stabilizers, solubility enhancers, and other suchagents, such as for example, sodium acetate, sorbitan monolaurate,triethanolamine oleate and cyclodextrins.

In an embodiment of the invention, pharmaceutically acceptable carriersused in parenteral preparations include aqueous vehicles, nonaqueousvehicles, antimicrobial agents, isotonic agents, buffers, antioxidants,local anesthetics, suspending and dispersing agents, emulsifying agents,sequestering or chelating agents and other pharmaceutically acceptablesubstances.

Examples of aqueous vehicles include sodium chloride injection, RingersInjection, isotonic dextrose Injection, sterile water injection,dextrose and lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations may be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEEN-80). A sequestering or chelatingagent of metal ions includes EDTA (ethylenediaminetetraacetic acid) orEGTA (ethylene glycol tetraacetic acid). Pharmaceutical carriers mayalso include ethyl alcohol, polyethylene glycol and propylene glycol forwater miscible vehicles; and sodium hydroxide, hydrochloric acid, citricacid or lactic acid for pH adjustment.

In an embodiment of the invention, preparations for parenteraladministration can include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

The concentration of the agent of the invention, which is optionally inassociation with a further chemotherapeutic agent, can be adjusted sothat a dose provides an effective amount to produce the desiredpharmacological effect. As discussed herein, the exact dose depends, inpart, on the age, weight and condition of the patient or animal as isknown in the art.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained, is also contemplated herein.Briefly, an active agent is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, orethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The antibody or fragment diffuses through the outer polymeric membranein a release rate controlling step. The percentage of active agentcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the antibody orantigen-binding fragment, which is optionally in association with afurther chemotherapeutic agent, and the needs of the subject.

Agents set forth herein can be formulated into a sustained releaseformulation including liposomal formulations such as unilamellarvesicular (ULV) and multilamellar vesicular (MLV) liposomes.

Example 1

The present invention is intended to exemplify the present invention andnot to be a limitation thereof. Methods and compositions disclosed belowfall within the scope of the present invention.

In the majority of sporadic colorectal cancers and in FAP, tumorigenesisis initiated from mutations that arise in the APC tumor suppressor gene.In the APC^(Min/+) murine model, numerous adenomas develop in theintestines, closely resembling FAP. APC^(Min/Min) homozygotes die inutero, and APC^(Min/+) heterozygotes spontaneously lose their wildtypeallele during puberty and start developing polyps by week 10. Althoughthese mice do not develop invasive metastases, they eventually succumbto a fatal anemia through excessive intestinal bleeding.

Prior studies in APC^(Min/+) mice show that chronic intestinalinflammation precipitates as well as propagates tumor growth and thatTh17 cells are the responsible immune effector cells. Since chronicinflammation in the intestine predisposes to colorectal cancer, it issurprising that the role of local DCs has yet to be examined in modelssuch as APC^(Min/+). The remarkable capacity of these cells toorchestrate distinct immune responses is aided by a panoply ofenvironmental cues, which condition the cells to adopt specificphenotypes in different settings. DCs in gut-associated lymphoid tissueare of particular interest because they maintain tolerance to commensalflora as well as mount protective inflammatory responses in the face ofpathogen incursion.

The experiments described in this report indicate that small intestineLPDCs (SI-LPDCs) play a critical role in the inflammatory process thatunderlies tumor progression in the APC^(Min/+) mouse. Whereas SI-LPDCsin healthy control mice induce the formation of T_(Regs), APC^(Min/+)SI-LPDCs are reprogrammed to induce Th17 formation. A marked reductionin the intestine of the vitamin A metabolite, retinoic acid (RA), whichunder homeostatic conditions maintains the tolerogenic functions ofLPDCs, explains the reprogramming of these cells. Since we findidentical defects in RA metabolism in FAP tissue, the same mechanismthat drives inflammation in APC^(Min/) mice contributes to tumorformation in FAP.

Inflammatory DCs accumulate in the SI-LP of APC^(Min/+) mice. Weinitially compared the frequency of DCs in various tissues inAPC^(Min/+) mice and their WT littermate controls. DCs were identifiedas EpCAM⁻CD45⁺Lin⁻MHCII⁺CD11c⁺ and analyzed by flow cytometry at 10, 14and 18 weeks of age, time points that correspond to minimal (<30 polyps,<2 mm diameter), intermediate (30-60 polyps, 0.5-4 mm diameter) and latestage (>60 polyps, 1-6 mm diameter) disease, respectively. As shown inFIG. 1a-b , DCs accumulated in the SI-LP as disease progressed. In thesteady state, DCs in the gut environment are comprised of threephenotypically distinct populations—CD103⁺CD11b⁻, CD103⁺CD11b⁺ andCD103⁻CD11b⁺DCs¹⁶. CD103⁺CD11 b⁻ DCs are enriched in the Peyer'spatches, whereas CD103⁺CD11b⁺ and CD103⁻CD11b⁺ DCs are found mainly inthe LP¹⁶. Although there were some differences in the frequency ofsplenic DC subsets between APC^(Min/+) and WT control mice, nosignificant differences in the percentages of the three key subsets wereobserved in the mesenteric lymph nodes (mLN), Peyer's Patches (PP) andmost importantly, the SI-LP (FIG. 1c ). These analyses were performed atintermediate stage, as the APC^(Min/+) mice lose their Peyer's Patchesby late stage disease, accompanying other developmental abnormalitiessuch as early thymic involution, splenomegaly and lymphodepletion.

We considered the possibility that the DCs accumulating in the SI-LP ofAPC^(Min/+) mice may be responsible for generating the altered CD4⁺ Tcell response in the gut demonstrated in previous studies. Nosignificant changes in the expression of costimulatory molecules (CD40,CD80, CD83, CD86 or PDL1) were seen on these cells (FIG. 1d ).Nonetheless, when stimulated with a panel of TLR agonists, purifiedSI-LPDCs from APC^(Min/+) mice, especially those with late-stagedisease, secreted much larger amounts of the pro-inflammatory cytokinesTNFα, IL-6 and IL-12p40 compared to SI-LPDCs from WT mice (FIG. 1e ).

APC^(Min/+) SI-LPDCs are impaired in their ability to induce T_(Regs)and instead promote Th17 formation. To address whether APC^(Min/+)SI-LPDCs are able to induce Foxp3⁺ T_(Regs) de novo, we culturedCFSE-labeled naïve CD4⁺CD62L⁺Foxp3⁻ T cells from OT-II TCR transgenicmice with purified DCs in the presence of TGFβ and the ovalbumin₃₂₃₋₃₃₉peptide. After 5 days of co-culture, the frequency of Foxp3⁺CD4⁺ T cellswas determined. The results show that splenic DCs from both genotypesinduced Foxp3⁺ cells weakly, while mLN/PP DCs were more potent inducersof Foxp3⁺ cells, consistent with previous studies. Interestingly,APC^(Min/+) SI-LPDCs were impaired in their capacity to induce Foxp3⁺cells compared to WT controls (FIG. 2a , FIG. 7a ). This impairment wasobserved beginning at intermediate stage and became more apparent atlate stage (FIG. 7c ). Similar results were obtained across a 25 foldrange of peptide concentration (FIG. 2b , FIG. 7b ).

When we measured the levels of key immunomodulatory cytokines in thesupernatants of these DC-T cell co-cultures, the results revealed astriking six-fold reduction in IL-10 generated in APC^(Min/+) LPDCco-cultures compared to the WT control cultures (FIG. 2e ). Moreover,there was a concomitant and similarly dramatic increase in IL-17A (FIG.20, consistent with the documented role of Th17 cells in adenomadevelopment. Intracellular staining of the T cells in the APC^(Min/+)co-cultures confirmed the presence of IL-17A producing cells and areduction of IL-10 producing cells.

As CD103⁺ SI-LPDCs are the main cells responsible for generating immunetolerance in the intestinal environment, we sorted SI-LPDCs into CD103⁺and CD103⁻ subsets to determine which subset accounted for the observedimpairment in Foxp3 induction. APC^(Min/+) CD103⁺ SI-LPDCs werefour-fold less able to induce Foxp3⁺ T cells compared to their WTcounterparts (FIG. 2c ). In contrast, CD103⁻ SI-LPDCs from bothgenotypes were equally poor in inducing Foxp3 expression (FIG. 2c ),consistent with previous studies. In addition, co-cultures ofAPC^(Min/+) CD103⁺ SI-LPDCs and T cells, but not CD103⁻ LPDC-T cellco-cultures, contained large amounts of IL-17A. These results show thatthe CD103⁺ subset of SI-LPDCs in APC^(Min/) mice is not only responsiblefor defective induction of T_(Regs), but also for the generation of Th17cells.

Retinoic acid (RA) reverses the inflammatory phenotype of APC^(Min/+)LPDCs. The unique capacity of the CD103⁺ SI-LPDC subset to store andmetabolize vitamin A into RA explains the specialized role of thesecells in generating and maintaining intestinal tolerance. Wehypothesized that the cytokine profile of the pro-inflammatoryAPC^(Min/+) SI-LPDCs could be modulated by changes in the local RAconcentration. To address this possibility we added RA or the RAreceptor antagonist, LE540, to the LPDC-T cell co-cultures and assessedthe induction of Foxp3⁺ T cells. LE540 abrogated Foxp3 induction in bothWT and APC^(Min/+) SI-LPDC co-cultures (FIG. 2d ), whereas RA enhancedFoxp3 induction in the APC^(Min/+) SI-LPDC co-cultures to the levelsseen in WT co-cultures (FIG. 2d ). Addition of RA also stronglyinhibited IL17A production in the APC^(Min/+) co-cultures. Since Th17differentiation requires IL-6 in conjunction with TGFβ, and since noexogenous IL-6 was added to our cultures, we postulated that theAPC^(Min/+) LPDC co-culture supernatants may contain IL-6. This wasconfirmed (FIG. 2g ). RA also potently inhibited IL-10 production inboth WT and APC^(Min/+) co-cultures, consistent with a previous study.

Loss of RA is linked to both reduced expression of retinaldehydedehydrogenases (Raldh) and defective regulation of the transcriptionalco-repressor C-terminal binding protein 1 (Ctbp1). Vitamin A, orretinol, when absorbed in the intestine, is either converted to astorage form or metabolized to RA. In the latter, retinol is catalyzedto retinal by two families of enzymes, the alcohol dehydrogenases (ADHs)and the short-chain dehydrogenase reductases (RDH), and subsequentlyoxidized to bioactive RA by the Raldh enzymes. To ascertain whetherAPC^(Min/+) LPDCs are able to produce RA, we used RT-qPCR to measure theexpression of several key Raldh enzymes in these cells. In line withprevious studies, Raldh expression in the SI-LP of both WT andAPC^(Min/+) mice was consistently higher than control splenic DCs (FIG.3b ). At early stage, APC^(Min/+) SI-LPDCs expressed equal or higherlevels of Raldh1a1 and Raldh1a2 compared to WT SI-LPDCs (FIG. 3a ).However, as the disease progressed in APC^(Min/+) mice, expression ofthese enzymes, especially Raldh1a1, declined markedly.

Recent studies have shown that RA in the gut induces Raldh expression.Since IECs constitutively express these retinol-metabolizing enzymes andcan efficiently generate RA, we postulated that defective RA productionin the IECs might help explain the loss of Raldh expression observed inthe APC^(Min/+) LPDCs. As shown in FIG. 3b , Raldh1a1 and Raldh1a2expression in sorted CD45⁻EpCAM⁺ IECs did not change significantlythrough early and intermediate stage disease. At late stage, however,APC^(Min/+) IECs exhibited approximately five- and two-fold reduction inthe expression of Raldh1a1 and Raldh1a2, respectively, compared to WTIECs. Of note, compared to WT IEC, ADH class I and II expression inAPC^(Min/+) IEC increased at intermediate stage and decreased atsubsequent late stage (FIG. 9). Taken together, these data show thatboth APC^(Min/+) SI-LPDCs and IECs lose expression of the criticalRaldh1a1 and Raldh1a2 enzymes by late stage disease, and have a reducedRA-production capacity compared to their WT counterparts.

To confirm that the changes observed at the transcript level of theseenzymes correlated with a loss of RA in the local tumor milieu, weutilized tandem mass spectrometry to quantitatively profile endogenousretinoids in late stage APC^(Min/+) duodenum, jejunum, ileum, colon andeye. As shown in FIG. 5i , RA levels in APC^(Min/+) tissues werediminished compared to WT controls, with the difference reachingstatistical significance in the ileum, where there is the highestfrequency of polyps, and in the colon. Although retinol was reduced inthe APC^(Min/+) ileum, tissues from both genotypes had comparableamounts retinyl esters, indicating that the deficit in RA is not due toa lack of substrate (FIG. 10).

Previous studies have demonstrated that the APC protein can directlybind to Ctbp1 in both Drosophila melanogaster and human cell lines.APC-mutant zebrafish were shown to express abnormally high levels ofCtbp1, which correlated with low levels of the intestinal retinoldehydrogenase Rdh11 and intestinal differentiation defects. Moreover,reintroduction of the APC protein into human colon carcinoma cell linesled to a proteasome-dependent destruction of Ctbp1, concomitant withincreased expression of the retinol dehydrogenase, DHRS9. To determineif the loss of RA we observed in the APC^(Min/+) gut may be due toupregulation of Ctbp1, we assessed Ctbp1 transcript levels in SI-LPDCsand IECs. A significant elevation of Ctbp1 was seen in APC^(Min/+) IECscompared to WT IECs beginning at intermediate stage disease (FIG. 3c ).Surprisingly, there was a parallel accumulation of Ctbp1 in theAPC^(Min/+) SI-LPDCs as well. These findings suggest that along withloss of Raldh expression, elevated levels in Ctbp1 in both cell typesmay suppress intestinal retinol dehydrogenase expression, therebycontributing to the loss of RA observed in the tumor milieu.

FAP adenomas exhibit the same inflammatory changes and abnormalities inRA metabolism as seen in APC^(Min/+) mice. To assess whether ourfindings in the APC^(Min/+) model are predictive of the human condition,we used immunohistochemistry and immunofluorescence to analyze thecolonic polyps of FAP patients. For comparison, we analyzed biopsies ofnormal colon as well as sessile serrated polyps (SSP), the latterserving as a source of non-APC mutated adenomas. IL17A expression wasstrikingly elevated in FAP adenomas compared to adjacent uninvolvedcolon, normal colon or SSP (FIG. 4a ). Staining of the same tissues withan isotype control antibody is shown in FIG. 11. These findings indicatethat the inflammatory environment in FAP polyps is similar to that seenin the APC^(Min/+) mouse model.

We next examined the tissues for expression of key vitamin Ametabolizing enzymes and the proteins that modulate them. Consistentwith published reports, cytoplasmic β-catenin was expressed at higherlevels in FAP adenomas compared to adjacent uninvolved tissue and toSSP. Interestingly, however, β-catenin was not expressed by cellsexpressing dendritic cell-specifiic Intercellular AdhesionMolecule-3-Grabbing Non-integrin (DC-SIGN) in the tissues tested (FIG.4b ). Ctbp1, the transcription factor that negatively regulatesintestinal RDHs, was present at much higher concentrationin FAP IECsthan in the IECs of normal or SSP (FIG. 5c ), consistent with previousreports, Remarkably, Raldh1a1 and Raldh1a2 expression was almostcompletely abolished in IECs of FAP adenoma tissue and not SSP (FIG.5d-e ), validating our findings in the APC^(Min/+) mouse. Finally, theRA catabolizing enzyme CYP26A1 was markedly upregulated in the epithelia(FIG. 5f ) as has been reported. Since FAP adenomas contain little RALDHactivity (possibly due to excess Ctbp1), but excessive CYP26A1, the netresult is likely a dearth of RA in the local tumor milieu.

Polyposis is exacerbated by a vitamin A deficient (VAD) diet andameliorated by Liarozole, a CYP26A1 inhibitor. Mice fed a vitaminA-deficient diet have dramatically reduced DC Raldh expression. Giventhe likelihood that diminished RA explains the pro-inflammatory profileof SI-LPDCs in APC^(Min/+) mice, we postulated that prolonged vitaminA-deficiency would intensify Raldh loss, drive inflammation andaccelerate polyp development. To test this hypothesis, groups of WT andAPC^(Min/+) mice were placed on a diet containing no vitamin A for 10weeks, beginning at 8-10 weeks of age, after which they were returned toa normal rodent chow diet. Groups of mice treated with Celecoxib (acyclooxygenase-2 inhibitor) and Rosiglitazone (a peroxisomeproliferator-activated receptor-γ agonist), were included as positiveand negative controls, respectively. These compounds were added to“base” mouse chow which contained 4 IU vitamin A/g. Disease developmentand severity were monitored on the basis of survival, changes in bodyweight, hematocrit and polyp count at the point of euthanasia.

WT mice on each diet gained weight comparably (FIG. 12) and did notdevelop polyps. APC^(Min/+) mice on the base diet survived throughoutthe 10-week period of disease monitoring, but all succumbed by week 24(FIG. 5a ). Compared to mice on the base diet APC^(Min/+) mice treatedwith Celecoxib had markedly prolonged survival, while mice that receivedRosiglitazone had reduced survival consistent with previous reports(FIG. 5a ). Interestingly, VAD diet-fed APC^(Min/+) mice hadparticularly aggressive disease and succumbed even more rapidly thanmice on Rosiglitazone (FIG. 5c-d ). The body weights of APC^(Min/+) miceplaced on base or VAD diets, or treated with Rosiglitazone, decreasedcomparably, while those treated with Celecoxib gained weight steadily(FIG. 5b ).

Since decreasing RA in the intestinal environment exacerbates disease inAPC^(Min/+) mice, we wanted to test the hypothesis that increasingintestinal RA would ameliorate disease. Oral administration of RA wasnot feasible, since it is known to stimulate further polyp growth inAPC^(Min/+) mice due to the induction of CYP26A1 and consequent loss ofRA. Instead, we sought to increase intestinal RA by preventing itsbreakdown with a CYP26A1 inhibitor, Liarozole, which was added to thebase diet of 8 week old mice for a period of 10 weeks, as above.Remarkably, the Liarozole-treated mice exhibited a striking reduction inthe number of polyps as well as a signficant increase in body weight,although their hematocrits deteriorated to the same extent as untreatedcontrols (FIG. 6a-d ). To confirm that Liarozole mediated its effects byincreasing local RA concentration in the intestine, we measured retinoidlevels in the treated mice. As shown in FIG. 6e , RA levels returned tonormal in Liarozole treated mice, especially in the ilium.

To assess whether the Liarozole mediated increase in intestinal RAinfluenced immune outcome by ‘re-reprogramming’ the pro-inflammatoryAPC^(Min/+) SI-LPDCs, SI-LPDCs from Liarozole-treated mice were isolatedand tested functionally. The results show that APC^(Min/+) SI-LPDCs fromLiarozole-treated miceno longer produced substantial amountsofpro-inflammatory cytokines when stimulated with TLR agonists (FIG. 60,and instead of inducing Th17 formation, promoted the formation of IL-10secreting Foxp3+ Tregs (FIG. 6g-h ) at levels similar to wildtype mice.Taken together, these findings show that whereas exacerbating RAdeficiency in the intestine accelerates disease, restoring RA reversesthe reprogramming of SI-LPDCs, thereby preventing deleterious Th17responses and ameliorating disease.

The role of intestinal inflammation in the development of adenomas inthe APC^(Min/+) model of spontaneous neoplasia has been well documented.However, the few immunological studies performed on APC^(Min/+) mice andrelated APC mutation models have focused almost exclusively on altered Tcell responses, with no attention directed at the underlying cause ofthese changes. Our findings indicate that as disease progresses, thereis an accumulation of pro-inflammatory DCs in the SI-LP that induce theformation of Th17 cells. This stands in dramatic contrast to theSI-LPDCs of healthy mice, which do not promote inflammation but insteadmaintain immune tolerance through the generation of T_(Regs). CD103+LPDCs, believed to be responsible for tolerance induction in theintestine, were present in greater numbers in APC^(Min/+) than healthycontrol mice, but secreted pro-inflammatory cytokines and induced theformation of Th17 cells rather than T_(Regs). The induction of Th17cells by APC^(Min/+) SI-LPDCs likely represents a critical control pointin shaping the inflammatory milieu that drives tumor growth, andexplains the predominance of IL-17 in the inflamed APC^(Min/+)intestine.

Since SI-LPDCs play such an important role in tumor-promotinginflammation, identifying the mechanism that induces their unusualphenotype in APC^(Min/+) mice is key to understanding how theinflammatory cascade is triggered. We hypothesized that these cellscould have undergone reprogramming in response to one or more factors inthe tumor microenvironment. Our efforts focused on RA because of thewell documented role of this molecule in maintaining a tolerant state inthe intestine. Addition of RA to the T_(Reg) induction cultures not onlyenhanced the capacity of APC^(Min/+) SI-LPDCs to induce Foxp3+ T cellsbut also prevented the induction of Th17 cells. Conversely, addition ofthe RAR antagonist, LE540, resulted in further diminution of Foxp3induction and augmented the production of IL17A by more than five-fold.

The absolute amount of RA in the intestines of mice with APC mutationshas not been reported previously, likely due to the technical difficultyof carrying out such measurements. By preventing exposure of tissues towhite light, which rapidly degrades retinoids, and utilizing massspectroscopy to measure RA, we could achieve accurate RA quantitation.Our results revealed highly significant reductions of RA in the ileumand colon of APC^(Min/+) mice, but not in other sites. Although some RAremained in the intestines, the reductions seen are nonethelessnoteworthy, as retinol bound to its specific retinol-binding protein(RBP) is strictly regulated and maintained in plasma at about 2 μMdespite daily fluctuations in dietary intake of vitamin A. Only insituations of severe, prolonged vitamin A-deficiency, where stores ofretinyl esters in the liver are depleted, is there a drop in plasmaconcentration of retinol-RBP, and by association, RA.

The loss of RA in the tumor milieu appears to be due to both diminishedsynthesis and excessive breakdown of this molecule. We found that thecritical Raldh enzymes that control RA production decline as diseaseprogresses, and this this is likely explained, in part, by theoverexpression of Ctbp1, which suppresses Rdh., Ctbp1 is normallydegraded by APC, but accumulates in the absence of functional APC. Ourimmunofluorescence data confirmed a marked accumulation of Ctbp1 in FAPIECs. Inactivation of the APC gene also results in constitutiveexpression of f3-catenin, which upregulates the major RA catabolicenzyme, CYP26A1. Indeed, the CYP26A1 transcript has been found to beupregulated in whole tissue isolated from both APC^(Min/+) and FAPadenomas, sporadic colorectal carcinomas, and the intestine ofAPC-mutant zebrafish embryos. Here we validate these observations,specifically identifying epithelial cells as one of perhaps several celltypes in FAP colon displaying CYP26A1 upregulation. Finally,prostaglandin E2 (PGE2), which has been reported to inhibit Raldh1a2expression in DCs, may contribute to the loss of RA. It is wellestablished that FAP and APC^(Min/+) epithelium exhibit constitutivelyhigh Cox2 expression. In addition, we found that the Cox2 transcript isoverexpressed in late stage APC^(Min/+) SI-LPDCs compared to their WTcounterparts (FIG. 13). This finding is consistent with the possibilitythat Cox2-overexpression and abundance of PGE2 in the FAP colon may leadto suppression of Raldh1a2 in both DCs and epithelia. Taken together,our results point to at least 3 mechanisms that cooperate to suppressthe RA levels in the tumor milieu of APC^(Min/+) mice. Moreover, thesedata indicate that the APC mutation underlying malignant transformationof intestinal epithelia is directly linked to the immune defect drivingtumor growth. We summarize our key findings and others in anillustration depicted in FIG. 6 d.

Importantly, human FAP adenoma tissue exhibited several of the samedefects that we found in the APC^(Min/+) mouse intestine. Not only wasthere a marked elevation in IL17 expression, signifying Th17 driveninflammation, but in addition we observed concomitant Raldhdownregulation and CYP26A1 upregulation in colonic epithelial cells.Defective RA production combined with enhanced RA breakdown wouldproduce a deficit of similar magnitude to that seen in APC^(Min/+) mice.

To investigate the role of RA in disease development and progression, wesought to alter RA concentration in the intestine in vivo, by removingor administering molecules in the diet that are known to affect RAproduction or breakdown. Our results revealed that a diet deficient inVitamin A exacerbates disease, while the CYP26A1 inhibitor, Liarozole,ameliorates disease as indicated by a dramatic reduction in the numberof intestinal as well as steady weight gain.

Since increasing intestinal RA proved highly efficacious in APC^(Min/+)mice, doing so in patients with APC-mutation associated bowel diseasemay be initiated. Small molecule agonists of Rdhs and Raldhs, orinhibitors of CYP26 such as Liarozole, are logical candidates. Indeed,Liarozole has been evaluated in clinical trials for diseases unrelatedto colorectal cancer and is apparently well-tolerated. Maintainingsufficient RA in the intestinal environment could therefore, reverseinflammation and reduce tumor burden, as we observed in APC^(Min/+)mice.

Materials and Methods

Mice. Breeding pairs of APC^(Min/+) male and WT C57BL/6 female mice werepurchased from The Jackson Laboratory and bred on-site. OT-II TCRtransgenic Rag^(−/−) mice were purchased from Taconic. TNF^(ΔARE/+) micewere kindly provided by Dr. Frank Jirik of the University of Calgary,Canada. All mice were housed in an American Association for theAccreditation of Laboratory Animal Careaccredited animal facility,maintained in pathogen-free conditions on a standard rodent chow adlibitum unless otherwise stated.

Isolation of DCs. Epithelial cells from the small intestine were washedin PBS with vigorous stirring and the remaining intestinal piecesdigested twice with Type VIII Collagenase (Sigma-C2139) and DNase I(Sigma). Spleen and mesenteric lymph nodes were digested withcollagenase Type IV (Worthington, 200 U/ml). For purification of DCs,cells were stained with monoclonal antibodies (Biolegend) againstCD45.2, CD49b, CD3e, CD19, CD11c, MHC II, EpCAM and propidium iodide(PI), and sorted using a FACS Aria (BD Biosciences). In some cases, DCswere additionally sorted using anti-CD103.

Flow Cytometric analysis. Isolated small intestine lamina propria(SI-LP) cells, splenocytes and mesenteric lymph node cells wereresuspended in 1% BSA in PBS (FACS buffer). After Fc blockade withanti-FcγRIII/II (BD Biosciences), cells were stained with Live/Dead Blue(Invitrogen) or PI, and monoclonal antibodies (all Biolegend) againstCD40, CD80, CD83, CD86, PDL1, Thy1.2 and CD4. Intracellular Foxp3 andcytokines were stained using antibodies against Foxp3, IL10, IL6, IL17A(all eBioscience) per manufacturer's instructions. Flow cytometricanalysis was performed on a LSRII flow cytometer (BD Biosciences).

T cell differentiation assay. 2×10⁴ sorted CD11c^(high) MHCII+ DCs wereco-cultured with 1×10⁵ MACS-enriched CD4+CD62L+Foxp3− naïve T cells fromthe spleen and lymph nodes of OT-II TCR-transgenic mice, along withOVA₃₂₃₋₃₃₉ peptide (New England Peptide) and 10 ng/ml recombinant humanTGF-β1 (Peprotech). On day 5, cells were harvested and analyzed forintracellular Foxp3 or intracellular cytokines. In some experiments,cells were restimulated on day 4 with 1 μg/ml each of plate-boundanti-CD3 and anti-CD28 (BD Biosciences), with or without Brefeldin A for18 hr. Where indicated, 10 nM all-trans RA (Sigma), or 1 μM LE540 (WakoChemicals) was added to culture wells.

ELISA. Purified SI-LP DCs were stimulated for 48 hr with Toll-likereceptor agonists—1 μg/ml Pam3Csk4, 10 μg/ml Poly I:C, 10 μg/ml LPS, 1μg/ml flagellin, 10 μg/ml R848, 10 μg/ml CpG 2336 (Invivogen).Supernatants from these experiments were assayed for IL-6, TNFα,IL12-p40 and those from were DC-T cell co-cultures were assayed forIL-6, IL-10 and IL17A, performed according to manufacturer'sinstructions (eBioscience).

Quantitation of gene expression using real-time PCR. Total RNA frompurified LP DCs, splenic DCs and epithelial cells was extracted usingRNAeasy kits (Qiagen), and the DNase-treated total RNA wasreverse-transcribed using High-Capacity Reverse Transcription Kit(Applied Biosystems) according to manufacturer's instructions. Geneexpression of ADH class 1-III, Raldh1a1-3, Ctbp1, and Cox2 wasdetermined by quantitative PCR with Power SYBR Green PCR Master Mix(Applied Biosystems) per manufacturer's instructions using a 7900HTreal-time PCR instrument (Applied Biosystems). Ubiquitin levels weremeasured in a separate reaction and used to normalize the data.

Quantitation of tissue retinoids. Retinyl esters (RE), all-transretinol, and all-trans retinoic acid (RA) were extracted from theduodenum, jejunum, ileum, colon and eye using procedures to thosedescribed previously. RA was quantified by LC/MS/MS with atmosphericpressure chemical ionization. RE and retinol were quantified by HPLC.Tissues were harvested and retinoids handled under yellow light usingonly glass laboratory equipment. Results were normalized to per gramtissue weight, or to control groups as fold-change.

Histology.

Immunohistochemistry.

Formalin-fixed, paraffin-embedded, 5 μM-thick, human tissue sectionswere stained with the primary antibody rabbit IL17A (Protein Tech Group(PTG) 13082-1-AP), and the secondary rabbit-HRP polymer (Dako Envision).Antigen retrieval was performed using Human Diva Decloaker citratebuffer (Biocare Medical).

Immunofluorescence.

Primary antibodies, all from PTG and rabbit anti-human unless otherwisenoted, were mouse DC-SIGN (Dendritics DDX0202), β-catenin (51067-2-AP),Ctbp1 (Human Protein Atlas (HPA) HPA018987), Raldh1a1 (15910-1-AP),Raldh1a2 (HPA010022), CYP26A1 (HPA C6498). Chicken secondary antibodiesinclude anti-rabbit Alexa647 and anti-mouse Alexa488 (Invitrogen). 11normal colon, 8 FAP adenoma, 2 FAP adenocarcinoma and 4 sessile serratedpolyp patients were analyzed. Images were collected using a Leica DM2500confocal laser scanning microscope, and analyzed using the LAS AFsoftware.

Drug Treatment.

1500 ppm of Celecoxib (Celebrex, Pfizer), 100 ppm of Rosiglitazone(Avandia, Glaxo Smith Kline), or 40 ppm of Liarozole (TocrisPharmaceuticals) was incorporated into a base diet (4 IU/g of vitamin A)by Research Diets. A vitamin A deficient (0 IU/g) diet (VAD) was alsostudied. Hematocrits were measured every 2 weeks, while weights and DAIwere measured every week. At the point of euthanasia, polyps wereenumerated and measured using a stereomicroscope at 10× magnification,from the gastro-duodenual junction to the ceco-colic junction.

Statistics.

An unpaired student's t test (2-tailed) with a 95% confidence intervalwas performed in Prism (Graphpad) in all experiments unless otherwisestated. Kaplan-Meier survival curves were analyzed with the log-ranktest. Differences of DAI in the drug treatment studies were analyzedusing the Wlcoxon Rank-Sum test in R. Where needed, mean±SEM wasrepresented on graphs. P<0.05=*; p<0.001=**; p<0.0001=***.

Example 2 Drug Treatment of Inflammatory Bowel Disease

Inflammatory DCs that infiltrate the GALT during autoimmune colitisappear to amplify Th17 responses that exacerbate intestinal pathology,similar to our observations in the APC^(Min/+) model of intestinalcancer. To address whether the loss of local RA as observed in ourstudies extended beyond APC-associated GI cancers, we quantifiedendogenous tissue retinoids as before in TNF^(ΔARE/+) andazoxymethane/dextran-sodium-sulphate (AOM/DSS)-treated mice. A model ofCrohn's disease that exhibit chronic ileitis, TNF^(ΔARE/+) mice providea model to examine whether there was a similar RA deficiency in cases ofgeneralized chronic intestinal inflammation. The AOM/DSS carcinogen andmucosal injury-induced model of colorectal carcinogenesis providedanother model of colorectal cancer, which is chemically-induced insteadof spontaneously-arising, like the APC^(Min/+). Intriguingly, comparedto WT tissue, we observed a marked reduction in RA in both the ileum andcolon of TNF^(ΔARE/+) and AOM/DSS mice, corresponding to the verylocations where inflammation and carcinoma was occurring respectively.

Taken together, an RA deficiency can occur in generalized settings ofchronic intestinal inflammation and in colorectal cancer, suggestingthat the beneficial effects of modulation of local intestinal RA levelsextends to generalized chronic inflammation in the gut.

As with many other hormones and vitamins that can be dysregulated indisease, reinstating RA to the appropriate dynamic range may be key. RAhas a short elimination half-life in vivo and in cultured cells just 6-7hours. Retinol bound to its specific retinol-binding protein in plasmais strictly regulated and maintained at about 2 μM despite dailyfluctuations from dietary intake of vitamin A. Only in situations ofsevere vitamin A-deficiency where stores of retinyl esters in the liverare depleted, is there a drop in plasma concentration of retinol-RBP.Corresponding RA levels in plasma are in the range of 5-10 nM, more thantwo orders of magnitude lower than that of its substrate retinol,reinforcing the notion that retinol metabolism is tightly regulated andoften restricted to certain settings. Local RA concentrations in theAPC^(Min/+) ileum and colon may be reduced to a point where tolerance isbroken, but not to the point where compensatory mechanisms that maintaintolerance in severe RA deficiency starts to kick in; or to the pointthat Th17 cells cannot induce inflammation anymore.

We measured RA levels in Liarozole-treated APC^(Min/+) mice and observeda return to wildtype levels of RA in the ileum, so we validated thatLiarozole did indeed increase local concentrations of RA to homeostaticlevels, and not to supraphysiological levels.

Since decreasing RA in the intestinal environment exacerbates disease inAPC^(Min/+) mice, we tested the reciprocal hypothesis that increasingintestinal RA would ameliorate disease. RA was administeredintraperitoneally (i.p.) twice weekly for 6 weeks to APCMin/+ mice,using the same protocol that has been reported to attenuate ileitis in amouse model of Crohn's disease. Surprisingly, compared to controls, thisregimen did not improve disease outcome in terms of tumor frequency,body weight or hematocrit. Consistent with these findings, SI-LPDCsisolated from APC^(Min/+) mice that received RA i.p. retained theirproinflammatory phenotype and ability to induce the formation of Th17cells. A likely explanation for this apparent paradox is that RA i.p.did not increase RA levels in the ileum, due to persistent breakdown ofRA from upregulated CYP26A1 in APC^(Min/+) tissue. Indeed, direct RAsupplementation via the diet has been shown to stimulate tumor formationin the APC^(Min/+) model.

Given these results, we decided to try an alternative strategy toincrease intestinal RA by targeting the upregulated CYP26A1 withLiarozole, an inhibitor of this enzyme. Compared to untreated and RAi.p.-treated mice, Liarozole treated mice exhibited a striking reductionin tumor number in the jejunum and ileum (FIG. 14a ), as well as asubstantial increase in body weight (FIG. 14b ), and hematocrit (FIG.14c ). Importantly, Th17 cells were reduced to WT levels (FIG. 14e ),and SI-LPDCs from these mice failed to induce Th17 cells, insteadpromoting the formation of IL-10-secreting CD4+ T cells (FIG. 14f ).Moreover, RA in the ileum of Liarozole-treated mice was restored to WTlevels (FIG. 145d ), likely explaining the therapeutic effect.Consistent with this interpretation, treatment of APC^(Min/+) mice withTalarozole, a more potent and more specific CYP26A1 inhibitor thanLiarozole38, also ameliorated disease (FIG. 15).

Taken together, these findings indicate that whereas increasing the RAdeficit in the intestine exacerbates disease, restoring RA to normallevels reverses the pro-inflammatory phenotype of SI-LPDCs, attenuatesTh17-driven inflammation and ameliorates disease.

Example 3 Inflammatory DCs Accumulate in the SI-LP of APC^(Min/+) Miceand Promote Th17 Formation and Tumor Growth

The frequency of DCs in various tissues was compared in APC^(Min/+) miceand their WT littermate controls. DCs(PI-EpCAM⁻CD45⁺Lin⁻CD11^(chi)MHCII⁺) were analyzed at 10, 14 and 18weeks of age, which correspond to early-(<30 adenomas, <2 mm diameter),intermediate-(30-60 adenomas, 0.5-4 mm diameter) and late-stage (>60adenomas, 1-6 mm diameter) disease, respectively. DCs accumulated in theSI-LP as disease progressed and by intermediate stage the frequency ofSI-LPDCs was more than 3 times greater in APC^(Min/+) than WT mice.

In the steady state, DCs in the gut consist of three phenotypicallydistinct populations: CD103⁺CD11b⁻, CD103⁺CD11b⁺ and CD103⁻CD11b⁺ DCs.Although there were some differences in the frequencies of splenic DCsubsets between APC^(Min/+) and WT control mice, no significantdifferences in the percentages of the three main DC subsets wereobserved in the mesenteric lymph nodes (mLN)/Peyer's Patches (PP) orSI-LP. These analyses were performed at intermediate-stage, asAPC^(Min/+) mice lose their Peyer's Patches by late-stage disease.Further studies of SI-LPDCs from APC^(Min/+) mice revealed that,although their expression of costimulatory molecules was similar to thatof WT SI-LPDCs, they secreted much higher levels of the pro-inflammatorycytokines TNFα, IL-6 and IL-12p40 under basal conditions and in responseto a panel of Toll-like receptor agonists. Moreover, APC^(Min/+)SI-LPDCs induced fewer Foxp3⁺ TRegs in a conventional TReg inductionassay. This impairment was observed at intermediate-stage and becameeven more apparent at late-stage disease. Similar results were obtainedacross a 25-fold range of peptide concentrations. Splenic DCs from bothgenotypes weakly induced Foxp3⁺ T cells, while mLN/PP DCs were morepotent inducers of Foxp3⁺ T cells, consistent with previous studies.

Supernatants obtained from APC^(Min/+) SI-LPDC-T cell co-culturescontained a striking six-fold reduction in IL-10 compared to WT SI-LPDCco-cultures. Moreover, there was a concomitant and similarly dramaticincrease in IL-17A, a key cytokine involved in adenoma development.Since Th17 differentiation requires IL-6 in addition to TGFβ and noexogenous IL-6 was added to our cultures, we measured IL-6 in co-culturesupernatants and, as expected, there were larger amounts in APC^(Min/+)SI-LPDC co-cultures. Consistent with these findings, IL-23, known to beessential for the maintenance of Th17 cells, though not detectable byELISA, was expressed at higher levels in APC^(Min/+) SI-LPDCs comparedto WT cells in situ.

As CD103⁺ SI-LPDCs are the main cells responsible for generating immunetolerance in the intestinal environment, we sorted SI-LPDCs into CD103⁺and CD103⁻ subsets to assess the role of each subset in the observedimpairment in Foxp3 induction. APC^(Min/+) CD103⁺ SI-LPDCs werefour-fold less efficient at inducing Foxp3⁺ T cells compared to their WTcounterparts. In contrast, cD103⁻ SI-LPDCs from both genotypes wereequally poor at inducing Foxp3 expression. Both CD103⁻ and CD103⁺APC^(Min/+) SI-LPDCs induced more Th17 cells compared to the WT SILPDCsubsets. These results show that the CD103⁺ subset of SILPDCs inAPC^(Min/+) mice is responsible for the observed defect in TReginduction, and that both CD103⁻ and CD103⁺ SI-LPDCs likely contribute tothe Th17-skewed inflammation observed in these mice.

To directly assess the role of DCs in tumor progression, we generatedbone marrow (BM) chimeras to selectively and constitutively ablate DCsusing BM donor cells in which Diphtheria Toxin A (DTA) is activated inCD11c expressing cells. Chimeras generated with WT and APC^(Min+) BMwere used for comparison. Depletion of DCs resulted in a significantdecrease in total tumor number, due mainly to a marked tumor reductionin the ileum where the incidence of tumors in APC^(Min/+) mice ishighest, providing strong evidence that DCs are a key driver of tumordevelopment. In contrast, APC^(Min/+) mice reconstituted with WT BM hadsimilar numbers of tumors compared to control APC^(Min/+)BM-reconstituted APC^(Min/+) mice, suggesting that the intestinalenvironment of the APC^(Min+) host overrides any potential benefitafforded by a reconstituted WT immune system.

Example 4 Tissue Histology

Immunohistochemical and immunofluorescence staining of intestinalbiopsies from FAP and APC^(min) mice shows the presence of identicalmarkers of inflammation as well as abnormal RA metabolism, includingRaldh1a1, Raldh1a2 and CYP26A1. These findings support the view that theunderlying pathology in APC^(min) disease is similar to that of FAP.Tumors in the APC^(min) model are primarily in the small intestine,whereas the tumors in FAP are primarily in the colon. One reason forthis difference is that the mouse colon has very few DCs, while DCs areabundant in the human colon. Drugs that work in APC^(min) mice also workin FAP, providing additional evidence that the pathogenesis of diseaseis the same in the two species.

Patients with ulcerative colitis, a type of inflammatory bowel disease,are at greatly increased risk for developing colon cancer due to thepresence of chronic inflammation. Once regions of microscopic dysplasiaare identified in the colons of such patients, the colons are typicallyremoved because dysplasia almost always becomes cancer.

Based on staining data, the same abnormalities in retinoic acidmetabolism that were found in APC^(min) mice, patients with FAP and micewith experimentally induced colitis and Crohn's disease were present inthe dysplastic areas of patients with ulcerative colitis.

What is claimed is:
 1. A method of reducing polyp development and/ortumor burden in an individual who has a mutation in the adenomatouspolyposis coli (APC) gene, the method comprising: administering to theindividual an effective dose of an agent to reduce polyp developmentand/or tumor burden in said individual, wherein the agent increaseslocal concentration of retinoic acid (RA).
 2. The method of claim 1,wherein the agent is not RA.
 3. The method of claim 2, wherein the agentincreases local concentration of RA through modifying enzymatic pathwaysinvolved in RA metabolism.
 4. The method of claim 3, wherein the agentincreases activity of retinaldehyde dehydrogenase or retinoldehydrogenase in intestinal tissues.
 5. The method of claim 4, whereinthe agent inhibits activity of a retinoic acid catabolizing enzyme. 6.The method of claim 5, wherein the retinoic acid catabolizing enzyme isa protein of the CYP26 family.
 7. The method of claim 6, wherein theretinoic acid catabolizing enzyme is CYP26A1.
 8. The method of claim 7,wherein the agent comprises one or more of: liarozole, talarozole,ketoconazole,[S—(R*,R*)]—N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine(R116010), and(R)—N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine(R115866).
 9. The method of claim 8, wherein the agent is Liarozole orTalarozole.
 10. The method of claim 1, wherein the agent is Liarozole orTalarozole.
 11. The method of claim 1, wherein said administeringcomprises oral administration.
 12. The method of claim 1, wherein theindividual has familial adenomatous polyposis (FAP).
 13. The method ofclaim 3, wherein said administering comprises oral administration. 14.The method of claim 3, wherein the individual has familial adenomatouspolyposis (FAP).
 15. The method of claim 6, wherein said administeringcomprises oral administration.
 16. The method of claim 6, wherein theindividual has familial adenomatous polyposis (FAP).
 17. The method ofclaim 8, wherein said administering comprises oral administration. 18.The method of claim 8, wherein the individual has familial adenomatouspolyposis (FAP).
 19. The method of claim 10, wherein said administeringcomprises oral administration.
 20. The method of claim 10, wherein theindividual has familial adenomatous polyposis (FAP).